Novel avian astrovirus

ABSTRACT

The present invention relates to the fields of veterinary virology and immunology. In particular the invention relates to a novel avian Astrovirus; to antibodies or fragments thereof against the novel virus; to antigenic preparations, proteins, and DNA molecules of the novel avian Astrovirus; to vaccines of the novel virus or its antigenic preparations, protein, or DNA; to methods for the manufacture of such vaccines, and to diagnostic kits.

The present invention relates to the fields of veterinary virology andimmunology. In particular the invention relates to a novel avianAstrovirus; to antibodies or fragments thereof against the novel virus;to antigenic preparations, proteins, and DNA molecules of the novelavian Astrovirus; to vaccines of the novel virus or its antigenicpreparations, protein, or DNA; to methods for the manufacture of suchvaccines, and to diagnostic kits.

Astroviruses are small, round, non-enveloped viruses having a singlestranded RNA genome of positive sense. Most Astroviruses have beenassociated with causing some sort of enteritis, giving rise todiarrhoea, vomiting, malabsorbtion, general malaise, and growthretardation. Infection can be lethal in humans or animals that are lessresilient, such as young, old, or immunocompromised individuals. See:Matsui and Greenberg (in: Fields virology, 3^(rd) ed., 1996, ed.: B.Fields et al., ISBN: 781702534, chapter 26), and: Moser andSchultz-Cherry (2005, Viral Immunology, vol. 18, p. 4-10).

Also, the severity of Astrovirus induced pathology can vary resultingfrom differences in virulence between Astrovirus species or strainsthemselves. This has created the belief that some of the Astrovirusesact as secondary pathogens, which by themselves cause only asub-clinical disease, while full clinical symptoms of disease are onlymanifested when there is an additional cause of disease from anotherpathogen.

The mechanism by which immunity against Astrovirus infection is achievedis also not completely clear; virus-neutralising antibodies do seem toplay a part in defence and immunological memory, but are probably notthe only effectors of an immunerespons against Astrovirus infection.This is reviewed by M. Koci (2005, Viral Immunology, vol. 18, p. 11-16).

Astroviruses have been detected worldwide and were isolated from a widevariety of animals. In turn, amongst the Astrovirus species infecting acertain target animal, several serotypes have been described.Taxonomically the Astroviridae family is divided into two main genuses,the mammalian Astroviruses: Mamastrovirus, and the avian Astroviruses:Avastrovirus. At the moment the genus Avastrovirus comprises theformally recognized species: Avian Nepritis Virus 1 and 2 (ANV1, 2)(from chickens), and Turkey Astrovirus 1 and 2 (TAstV1, 2).

Several additional avian Astroviruses have been isolated from duck,chicken, ostrich and turkey. For instance, chicken astrovirus 2 (CAstV2)has been described by Baxendale and Mebatsion (2004, Avian pathology,vol. 33, p. 364-370; and in international patent application WO2004/027053), and Todd et al. described a CAstV3 (WO 2007/077464), whichis closely related to CAstV2. These Astroviruses have not yet beenformally classified, as reviewed by Koci and Schulz-Cherry (2002, AvianPathology vol. 31, p. 213-227).

Astroviruses are very stable in the environment, e.g. they can resistcommon lipid-solvents and detergents, have a wide pH tolerance, and arecomparatively thermostable. This, combined with their high infectivityand easy transmission by faecal-oral route, explains why Astroviruses orantibodies against them can be found in so many humans and animalsworldwide.

The classification and naming of these small RNA viruses has beensubject to frequent change, when updating was required to fit newexperimental data. For instance: the duck Astrovirus (DAstV) wasformerly classified as a Picorna virus, and was originally called duckhepatitis virus type II, as it causes hepatitis. Similarly, ANV1 wasformerly described as a Picornavirus, in particular an Enterovirus, butwas re-classified to the Avastroviruses (Imada et al., 2000, J. ofVirology, vol. 74, p. 8487-8493). ANV1 is also called: chickenAstrovirus 1 (CAstV1), and causes nephritis.

Re-classification of these viruses was called for upon the availabilityof new information on the sequence and organisation of their viralgenome. Particularly for Astroviridae, it was shown that physical,electron-microscopic, and biochemical characterisations often do notsuffice to differentiate the Astroviridae from other so-called “smallround viruses”, or “enterovirus-like viruses” such as Picorna-, Calici-,or Hepeviridae (Hepatitis E like viruses), or even from enveloped RNAviruses such as Reo-, Rota-, or Coronaviruses which cause diseasesymptoms much resembling those of Astroviruses. This is because theresults of such characterisations vary with the method and theconditions used. Therefore these are not consistent enough to make theright determination.

On the other hand, molecular biological characterisation such asgenotyping, in combination with immunologic characterisation is detailedand reproducible enough to assign a micro-organism to the propertaxonomic group (Van Regenmortel et al., 2000, in: Virus Taxonomy,7^(th) report of the ICTV, Academic press, New York). Therefore ideallysuch determinations are based on nucleotide sequencing, polymerase chainreaction (PCR) assays, and serological typing assays.

The RNA genome of the Astroviridae is roughly about 7 kb in size, with agenome organisation that is typical for this virus family, see: Jiang etal. (1993, PNAS-USA, vol. 90, p. 10539-10543) and Koci et al. (2000, J.of Virology, vol. 74, p. 6173-6177). According to current understanding,the genome incorporates three clearly recognisable open reading frames(ORF's), from 5′ to 3′:

-   ORF 1a: is about 3 kb in size, encodes a non-structural protein    having a serine protease motif.-   ORF 1b: is about 1.5 kb in size, partly overlaps with ORF 1a, and    encodes a non-structural RNA-dependent RNA polymerase.-   ORF 2: encodes the structural viral proteins, via a sub-genomic RNA    of about 2 kb. The expresssed polyprotein is probably cleaved into    smaller parts before viral assembly, and comprises a.o. the capsid-    or coat protein.

Of the three ORF's, ORF 1b has the most conserved nucleotide sequence ascompared among the Astrovirus nucleotide sequences that are publiclyavailable, and ORF 2 is the ORF most variable in sequence.

Some particular disease problems were observed between 1987 and today,in poultry operations in the Netherlands, Germany and United ArabEmirates, with chickens and turkeys suffering from diarrhoea, reductionin feed conversion and growth, as well as from problems with swellingand painfulness of the joints and tendons of the legs. These problemswere found in broiler-, breeder-, and layer type birds, of various ages,and at different farms having no apparent mutual connection.

Lameness was observed in birds in their first week of age, up untilseveral months of age. Animals presented swollen hock (ankle) joints andtendon-shafts of the lower leg, both overfilled with a clear, slightlyviscous liquid, and symptoms of arthritis. A reduction of the feedutilization and growth rate was observed in these birds, most likely asresult of the locomotory problems. In the worst cases the birds were nolonger able to move and thereby get feed or drink, resulting inmortality. These leg abnormalities were most prominent in the heavierbroiler-type birds, however the economic damage sustained wasconsiderable in all types of birds.

Even more impact had the diarrhoea that was observed, mainly in youngerbirds, but also in birds of egg-laying age. Symptoms varied fromindigestion to clear enteritis, and resulted in reduction of feedconversion; growth depresssion or even weight loss; drop in eggproduction (“egg-drop”); increase in the numbers of eggs of poorquality; and mortality.

On some farms the problems were observed over several years, resultingin sustained bad production figures without definite cause.

Upon post mortem investigations, in several instances body parts otherthan intestines or leg joints were also found to be affected, such asdisplayed by erosion of the gizzard or swelling of the liver.

To detect the cause for these symptoms, aspects of management and dietwere investigated but these could not be linked to the problems observedin these cases. Also pathogens were investigated that could beresponsible, in particular an infection by Reovirus, as this virus iswell known for causing malabsorbtion and joint problems. However, inspite of the use of a variety of assays, in many cases no Reovirus couldbe detected.

Several other pathogens were also investigated either routinely orspecifically for being known to cause similar problems, such as thebacteria: Salmonella, Mycoplasma, Haemophilus, and Pasteurella; and theviruses: Adenovirus, Infectious Bronchitis virus (IBV), NewcastleDisease virus (NDV), Infectious Bursal Disease virus (IBDV), Egg DropSyndrome (EDS), and Avian Influenza virus (AIV).

Up until today, no causative agent could be linked to the leg- andintestinal problems that were observed, in spite of the severity, theproblems for the animal's welfare, and the resulting bad economicperformance of these poultry operations.

Consequently a need exists to identify an agent connected to thedisease-symtoms observed and to provide vaccines and diagnostics, whichprovide prevention of the disease and identification of the causativeagent.

Surprisingly a new virus has been found in chickens and turkeyssuffering from intestinal and locomotory disease. The virus could beisolated from diseased birds of various ages, types, and origins, bothfrom joints and tendons as well as from various internal organs. Theisolated virus in turn induced similar symptoms of disease uponinfection of healthy test animals, and could be re-isolated from thediseased test animals, thereby fulfilling Koch's postulates.

The new virus can be used to develop diagnostic assays and vaccines todetect and to combat the virus and the disease problems it causes inpoultry, in particular disease of joints and tendons as well as of thegastro-intestinal tract.

The novel virus was analysed in a number of ways, and was surprisinglyidentified as an Astrovirus. Because of its prevalence in chickens andturkeys it is tentatively qualified as an avian Astrovirus.

Among many other characterisations, the fact that the new virus asdescribed herein is non-enveloped followed from the results of twoconsecutive extractions with chloroform, after which the extracted virussample still was able to cause a specific pathogenic effect inembryonated chicken SPF eggs.

The presence of an RNA genome in the new virus followed from the factthat useable nucleic acid for further analysis could only be obtainedafter RNA extraction and RT-PCR, not after DNA isolation and regularPCR.

Surprisingly, the closest (yet distinguishable) relatives were found tobe ANV1 type viruses, therefore the novel avian Astroviruses describedherein represent a novel group of avian nephritis viruses, tentativelyclassified as ANV3 viruses.

However the inventors have surprisingly found a number of features thatdistinguishes the novel avian Astroviruses described herein from ANV1and other (Astro-) viruses, and uniquely characterises the differentisolates of the novel avian Astrovirus as belonging to a novel andseparate group of its own.

The main characterising feature relates to the presence of a specificnucleotide sequence which is identifyable by nucleotide sequenceanalysis and by PCR assay.

Distinct pathologic symptoms induced upon infection of embryos were alsoobserved. Specific immunological differences were detectable byserotyping using VN- or IFT-assays. All these will be described belowwith experimental results and -details.

The invention relates to an isolated novel avian Astrovirus having anopen reading frame (ORF) 1a genomic region, characterised in that theORF1a of said avian Astrovirus, when compared to the ORF 1a of aviannephritis virus 1, contains an insert of 12 nucleotides, said insertbeing located in between nucleotides corresponding to the nucleotidesnumbered 2485 and 2486 of SEQ ID NO: 1.

Preferred “avian” organisms are poultry; more preferred avian organismsare selected from the group consisting of chicken, turkey, duck andgoose. Most preferred is chicken.

The term “Astrovirus” indicates the relationship of the group of newviruses according to the invention to viruses that are currentlydescribed and/or classified as belonging to the taxonomic family of theAstrovirideae. However, the skilled person will realise that suchtaxonomic classificaton may change over time as new insights could leadto reclassification into new or other taxonomic groups. However, as thisdoes not alter the characteristics of the virus according to theinvention but only its classification, such re-classified organismsremain to be within the scope of the invention.

In particular the invention encompasses all viruses sub-classified fromthe avian Astrovirus according to the invention into a sub-species,strain, isolate, genotype, serotype, variant or subtype and the like.

Therefore, for the invention, an “Astrovirus” is a virus having thecharacterising features of a virus classified as an Astrovirus. Suchcharacterising features relate e.g. to molecular, bio-chemical, andbiological features; for instance to having a positive sense singlestranded RNA genome comprising ORF's identifiable as 1a, 1b and 2; beinga non-enveloped virion of about 28-30 nm; and causing an enteritis.

The term “genomic region” is meant to indicate a part of the geneticmaterial of the novel avian Astrovirus according to the invention. Sucha region will consist of nucleic acid; in the case of the novel avianAstrovirus according to the invention, the nucleic acid is RNA.

The genome of the novel avian Astrovirus according to the inventioncomprises, as do all other Astrovirideae, regions recognisable as andcorresponding to ORF's 1a, 1b and 2.

However, it was surprisingly found that the novel avian Astrovirus hassome nucleotides in its ORF 1a genomic region which are not present inthe ORF 1a of ANV1 or of any other type of (avian) Astrovirus. Thesenucleotides are present in the form of a linear and continuous stretchof 12 nucleotides, and when compared to the ORF 1a sequence of ANV1represent an insert into this region. The 12 nucleotide “insert” waspresent in the ORF 1a of all the isolates tested of the group of novelavian Astroviruses described herein.

The location where the 12 nucleotide insert is present in the ORF 1a ofthe isolates of the novel avian Astroviruses described herein, isindicated by reference to the numbering as described for the genomicsequence of the reference ANV1 strain, which was described by Imada etal. (2000, J. of Virology, vol. 74, p. 8487-8493). The cDNA sequence ofthe genomic sequence of this ANV1 strain is also available from theinternet nucleotide-database of the American National Institutes ofHealth, known as Gen Bank, under accession number: AB033998, and isrepresented herein as SEQ ID NO: 1.

For all isolates of the novel avian Astroviruses according to theinvention, the 12 nucleotide insert in ORF 1a was located in betweennucleotides corresponding to the nucleotides numbered 2485 and 2486 ofSEQ ID NO: 1.

At present it is not known if, or how, this 12 nucleotide insert in ORF1a of the novel avian

Astrovirus according to the invention, which is not present in ORF 1a ofANV1, influences the behavior of this novel avian Astrovirus. Withoutbeing bound to any theory, the inventors assume that the fact that theinsert exist of 12 nucleotides, thus 4 triplets which keeps thetranslational frame of ORF1a intact, makes that the encoded 4 additionalamino acids are incorporated in the non-structural proteins that areexpressed from ORF 1a. It is very well possible that this influences theviruses' patho-biological behaviour, for instance its virulence and itscapacity to cause disease of the intestine as well as of legs, jointsand tendons.

Nevertheless, the fact that these 12 nucleotides are present in the ORF1a genomic region of the novel avian Astroviruses described herein, butnot in the ORF 1a of ANV1 or other (avian) Astroviruses, provides apositive genetic marker for this novel group of viruses.

As is well known in the art, a genetic marker is a feature of anorganisms' genetic material, situated at a certain genomic location,that is useful! for making a certain distinction or correlation, and isdetectable by technical means. When—as in this case—a feature is presentin a group to be identified, it is a “positive” genetic marker for thatgroup.

The nucleotide sequence of the 12 nucleotide insert that characterisesthe novel avian Astrovirus according to the invention, is not identicalfor all the isolates of the novel avian Astrovirus described herein,however size and location were identical. The consensus of thenucleotide sequence of the 12 nucleotide insert is:

5′-TCYGGDMARYYT-3′, represented herein as SEQ ID NO: 2.

Therefore, in a preferred embodiment the invention is characterised inthat the insert of 12 nucleotides has a nucleic acid sequence aspresented in SEQ ID NO: 2.

The skilled person will know that the sequence of SEQ ID NO: 2 is aso-called ‘denatured’ primer sequence, indicating that there arepositions (here: the Y, D, M, and R), where one of a number ofnucleotides can be present. The commonly used code for indicatingdenatured bases is the IUPAC code (published in Biochem. J., 1985, vol.229, p. 281-286), wherein: R=G or A; Y=T or C; M=A or C; K=G or T; S=Gor C; W=A or T; H=A, C, or T; B=G, T, or C; V=G, C, or A; D=G, A, or T;and N=G, A, T, or C.

For the novel avian Astrovirus described herein, the presence of thisdetectable genetic marker allows for the identification of the novelvirus and its novel group by detection for instance via DNA sequencingor PCR.

DNA sequencing is a well known technique; standard protocols andequipment for instance for high speed automated sequencing are widelyavailable. Most commonly such protocols employ PCR based“cycle-sequencing”, followed by high-definition electrophoresis.

For the nucleic acid sequencing of RNA viruses such as Astrovirideae,the nucleic acid to be investigated needs to be in the form of copy DNA(cDNA), as RNA sequencing is not yet feasible. For the preparation ofcDNA many standard protocols and commercial kits are available. Suchprotocols employ a reverse transcriptase enzyme. Commonly a primer isused to initiate the copying process.

An example of a suitable primer for the RT reaction is the DNAoligonucleotide presented herein as SEQ ID NO: 3, being: 5′-TCG WTS CTACYC-3′. The inventors refer to this primer as primer 17.

This primer hybridises to the far 3′ region of the genome of many avianAstroviruses, starting at the nucleotide corresponding to nucleotidenumber 6731 of SEQ ID NO: 1, and proceeding in reverse direction.

In FIG. 1 is displayed a multiple alignment of the cDNA sequence of asection of ORF 1a from a selected number of isolates of the avianAstroviruses according to the invention. These are aligned to thecorresponding part of the genome of an ANV1 reference virus, asrepresented in SEQ ID NO: 1. As is clearly visible in the boxed area,all isolates possess a 12 nucleotide region, which is not present in theANV1 ORF 1a. The SEQ ID NO's of the ORF 1a cDNA sequence of the isolatesof the avian Astrovirus according to the invention are listed in Table1.

Similarly, FIG. 2 displays a multiple alignment of the putative aminoacid sequences, as prepared by computer translation of the cDNAsequences listed in FIG. 1. As is visible in the boxed area, the proteinencoded by ORF 1a of the avian Astroviruses according to the inventioncontains 4 amino acids, that are not present in the protein encoded byORF 1a of ANV1. The SEQ ID NO's of the translated ORF 1a sequence of theisolates of the avian Astrovirus according to the invention are listedin Table 1.

With respect to the isolates of the avian Astroviruses according to theinvention: a large number of field isolates were collected over timefrom chickens and turkeys suffering from leg and/or intestinal diseaseas described above. Of these isolates many were analysed, which led tothe invention of the novel group of avian Astroviruses as describedherein. However, only a selection of all analysed isolates are presentedherein, to define the novel avian Astrovirus in the context of thenatural variation occurring in that group.

Table 1 presents a description of the isolates of the avian Astrovirusaccording to the invention, that are described herein. Also Table 1lists the SEQ ID NO's of the cDNA and putative protein sequences fromORFs 1a, 1b and 2 from a selection of isolates.

TABLE 1 Characteristics of a selection of isolates of the avianAstrovirus according to the invention; also SEQ ID NO's of sequencedcDNA regions (“nt”) and putative encoded proteins (“aa”). Donor animal:SEQ ID NO's Isolate number species type age tissue Orf 1a Orf 1b Orf 2161319 Chicken layer 36 w Trachea nt: 4 nt: 6 nt: 8 “Isolate 19” aa: 5aa: 7 aa: 9 637 Chicken broiler 12 d Tendon and nt: 10 hock-cartilageaa: 11 686 Chicken broiler 10 d Various organs nt: 12 aa: 13 714 Chickenbroiler 30 d Ceacal tonsils nt: 14 and pancreas aa: 15 715 Chickenbroiler 30 d Ceacal tonsils nt: 16 and pancreas aa: 17 1736 Chickenbroiler — Tendon and nt: 18 tibia aa: 19 2383 Turkey broiler — Joint andnt: 20 synovial fluid aa: 21 2388 Turkey broiler — Tendon and tibia nt:22 aa: 23 7279 Chicken — — Pancreas nt: 24 aa: 25 161317 Chicken layer36 w Trachea nt: 26 aa: 27 “nt” = nucleotide sequence; “aa” = amino acidsequence; “—” = not known

An isolate of the avian Astroviruses according to the invention can beobtained from birds suffering from intestinal or leg-problems by takinga sample from one or more organs, or from joints or tendons of the legs.The samples can be homogenised when required, and for instance be mixedwith an appropriate buffer solution, preferably containing antibiotics.

To amplify the viral titre of such isolates, different substrates can beused, such as avian cell-lines or embryonised avian eggs. When usingeggs, the suspension can be inoculated into and cultured in embryonatedavian eggs. Different inoculation routes are possible, such as on thechorio-allantoic membrane (CAM), or into the yolk sac, or the allantoicfluid cavity. Preferably, the first few rounds of inoculation are intothe yolk sac, and later ones (if required) can be into the allantoicfluid. About 5-7 days old, embryonated chicken SPF eggs can be used.Such techniques are all well known to persons skilled in the art, andSPF chicken eggs of appropriate quallity and age are commerciallyavailable, for instance from Spafas®, or Lohmann®.

After incubation for 2-7 days under appropriate conditions, the embryo,the CAM, or (preferably) the allantoic fluid can be harvested. Ifrequired further rounds of amplification on eggs can be performed, byinoculation into the yolk sac or the allantoic fluid. Finally allantoicfluid can be harvested containing high amounts of the avian Astrovirusaccording to the invention.

For PCR-sequencing of both strands of a representative part of ORF 1athe following primers can conveniently be used:

SEQ ID NO: 28: 5′-AAA GGK AAG ACD AAG ARR RAC MG-3′, and SEQ ID NO: 29:5′-TCG CCT TCT GGA AGG TCT TCA-3′.

The inventors refer to these sequencing-primers as primers F-II andR-II-3 respectively. SEQ ID NO: 28 hybridises starting at nucleotidescorresponding to nt 2171 and further of ANV1 (SEQ ID NO:1); SEQ ID NO:29 hybridises to nucleotides starting at nt 2640 of SEQ ID NO: 1, andproceding in reverse direction (see Table 4). This way the genome regionof ORF 1a of the avian Astrovirus according to the invention comprisingthe 12 nucleotides not present in ANV1, is covered by the part of ORF 1athat is sequenced.

For computer analyses of the DNA sequences determined, these are trimmedso that they do not contain the denatured sequences from the primersthemselves. Also the sequences compared need to be of equal length toachieve representative alignments. Also, the alignments are to be madeover the full length of the trimmed sequences. That means for instancein the case of using the primers SEQ ID NO: 28 and 29, that the sequencelength to be compared is about 400-425 nucleotides.

A convenient computer program for such manipulations and alignments isClone Manager™ (by: Scientific and Educational software™), alternativelyprograms are available via the internet: “ClustalW” for multiplealignments, and “Translate” for translations, both accessible atwww.expasy.org; or the “Blast” programs at www.ncbi.nlm.nih.gov.Preferably the default scoring parameters are employed.

cDNA sequencing using primers SEQ ID NO: 28 and 29 was done for manyisolates of the avian Astroviruses according to the invention. Aselection of the resulting, trimmed nucleotide sequences are presentedherein as in SEQ ID NO's: 4, 10, 12, 14, 16, 18, 20, 22, 24 and 26, asrepresented in Table 1.

These sequences were aligned to each other, using as reference thesequence of one isolate from a chicken, numbered 161319, to which theinventors refer as “isolate 19”. Also, several DNA sequences of thecorresponding parts of ORF 1a from other Astroviruses available inGenBank were aligned, for instance ANV1 (AB033998, SEQ ID NO: 1), turkeyAstrovirus 1 (TAstV1) (Y15936), turkey Astrovirus 2 (AF206663), sheepAstrovirus (Y15937), mink Astrovirus (AY179509), and human astrovirus(Z25771); between hyphens are the respective GenBank accession numbers.

Of these other Astrovirus ORF 1a sequences, only ANV1, in particular thepart of nt 2213-2607 from SEQ ID NO: 1, had a sequence identity that wassignificant, which means a nucleotide sequence identity percentage wellabove 50%. Of the remaining other Astroviruses, only TAstV1 (GenBankacc. nr: Y15936) had a detectable, but hardly significant sequenceidentity in the part of nucleotides nr. 2450-2856. This is representedin Table 2.

TABLE 2 List of percentage nucleotide sequence identity between SEQ IDNO: 4 (being a part of ORF 1a from isolate 19), and the sequence of thecorresponding part of ORF 1a from other isolates of the avian Astrovirusaccording to the invention, as well as from other Astroviruses.Isolate/virus % nucleotide sequence SEQ ID NO, or name identity to SEQID NO: 4 GenBank accession no. 637 90 SEQ ID NO: 10 686 88 SEQ ID NO: 12714 89 SEQ ID NO: 14 715 98 SEQ ID NO: 16 1736 89 SEQ ID NO: 18 2383 88SEQ ID NO: 20 2388 88 SEQ ID NO: 22 7279 89 SEQ ID NO: 24 161317 89 SEQID NO: 26 ANV1 80 SEQ ID NO: 1 (part: 2213-2607) GenBank: AB033998TAstV1 56 GenBank: Y15936 (part: 2450-2856) TAstV2 <50  GenBank:AF206663 (part: not detectable)

A dendrographic tree of the results of these nucleotide sequencealignments is presented in FIG. 3.

Thus, it was surprisingly found that the group of novel avianAstroviruses according to the invention stand apart from all otherAstroviruses, avian or mammalian, in that they share a nucleotidesequence identity of 88% or more when comparing a region of ORF 1a DNAsequences corresponding to SEQ ID NO: 4.

Therefore, in a preferred embodiment, the invention relates to the avianAstrovirus according to the invention, characterised in that the ORF 1aof said avian Astrovirus comprises a region having a nucleotide sequenceidentity of at least 88% with SEQ ID NO: 4.

More preferably, the invention relates to an avian Astrovirus accordingto the invention, characterised in that the ORF 1a of said avianAstrovirus comprises a region having a nucleotide sequence identity ofat least 89% with SEQ ID NO: 4, even more preferably 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100% identity, in that order of preference.

Next to SEQ ID NO: 4, any one of the partial ORF 1a nucleotide sequencesdescribed in SEQ ID NO's: 10, 12, 14, 16, 18, 20, 22, 24 and 26, mayserve to characterise the avian Astrovirus according to the invention infurther preferred embodiments.

Analogous to the multiple alignment of the nucleotide sequences from ORF1a as presented in Table 2, alignments can be made of the putative aminoacid sequences from the trimmed nucleic acid sequences of Table 2, theSEQ ID NO's: 4, 10, 12, 14, 16, 18, 20, 22, 24 and 26. The resultingamino acid sequences—translated by computer—are presented herein as SEQID NO's: 5, 11, 13, 15, 17, 19, 21, 23, 25, and 27. The referencesequence is SEQ ID NO: 5, the putative amino acid sequence of part ofORF 1a of Isolate 19. The alignment was also made using theAlign+program, aligning over the full length of SEQ ID NO: 5. Theresults are presented in Table 3.

TABLE 3 List of percentage amino acid sequence identity between SEQ IDNO: 5 (being a translation of a part of ORF 1a from isolate 19), and thesequence of the corresponding part of ORF 1a from other isolates of theavian Astroviruses according to the invention, as well as from ANV1 andTAstV2. Isolate/virus % amino acid sequence SEQ ID NO, or name identityto SEQ ID NO: 5 GenBank accession no. 637 97 SEQ ID NO: 11 686 97 SEQ IDNO: 13 714 97 SEQ ID NO: 15 715 99 SEQ ID NO: 17 1736 96 SEQ ID NO: 192383 94 SEQ ID NO: 21 2388 93 SEQ ID NO: 23 7279 96 SEQ ID NO: 25 16131797 SEQ ID NO: 27 ANV1 88 GenBank: BAA92848 (part: 734-864) TAstV2 <40 GenBank: Q9ILI6 (part: 810-930; hardly detectable)

The detailed amino acid alignment is represented in FIG. 2.

The result of this amino acid sequence analysis, shows an outcomecomparable to the results in Table 2: the amino acid sequences from theisolates of the avian Astrovirus according to the invention are morerelated amongst themselves, than when compared to other avianastroviruses. Identity percentages of the amino acid sequences of thevarious isolates of the avian Astrovirus according to the invention toSEQ ID NO: 5, are between 93 and 99%, while identity to ANV1 is less, at88%.

Therefore, in a preferred embodiment, the invention relates to an avianAstrovirus according to the invention, characterised in that the ORF 1aof said avian Astrovirus encodes an amino acid sequence comprising aregion having an amino acid sequence identity of at least 93% with SEQID NO: 5.

More preferably, the invention relates to an avian Astrovirus accordingto the invention, characterised in that the ORF 1a of said avianAstrovirus encodes an amino acid sequence comprising a region having anamino acid sequence identity of at least 94% with SEQ ID NO: 5, evenmore preferably 95, 96, 97, 98, 99, or 100% identity, in that order ofpreference.

Next to SEQ ID NO: 5, any one of the partial ORF 1a putativelytranslated nucleotide sequences described in SEQ ID NO's: 11, 13, 15,17, 19, 21, 23, 25, and 27, may serve to characterise the avianAstrovirus according to the invention in further preferred embodiments.

In addition to nucleotide sequencing and alignment, the positive geneticmarker of the avian Astroviruses according to the invention can beidentified by using a PCR test to detect the presence of the 12nucleotides in the ORF 1a region of the avian Astroviruses according tothe invention, which are not present in the ORF 1a of ANV1, for instancein samples from birds suffering from intestinal or locomotory disease.

Therefore, in a further preferred embodiment, the avian Astrovirusaccording to the invention is characterised in that from the ORF 1a ofsaid avian Astrovirus a PCR product of about 260 nucleotides can beproduced in a PCR-assay using a set of the primers represented in SEQ IDNO's: 30 and 31.

The PCR primers to be used are:

SEQ ID NO: 30: 5′-GTY CTY ACC GAR GAR GAR TAY C-3′SEQ ID NO: 31: 5′-AAD GTT ATY CTC CTA RGB TKH C-3′

The inventors refer to these primers as primers 29 and 30, respectively.

These primers hybridise to nucleotides corresponding to ANV1 (SEQ IDNO: 1) nucleotide number 2252 and further, and to nucleotide number 2499and proceeding in reverse.

The PCR product that was produced was visible on a gel as a band ofabout 260 nucleotides (this is including the length of the primersthemselves). This band is produced only when the starting materialcontains DNA comprising the ORF 1a region of the avian Astrovirusesaccording to the invention, as only these comprise the 12 nucleotideswhich are not present in ANV1 ORF 1a. A graphic representation of thespecific binding of primers SEQ ID NO's: 30 and 31, in relation to thelocation of the 12 nucleotide region is presented in FIG. 4.

In such PCR assays the specificity and the sensitivity is determined bythe specific PCR primers used, in particular by the primer's sequenceand length. Optimisation of selectivity and sensitivity can then beachieved by adapting the reaction- and cycling conditions, such as thetemperature and duration of the reaction steps, as is well known to askilled person.

These, and other molecular biological techniques are explained in greatdetail in standard text-books like Sambrook & Russell: “Molecularcloning: a laboratory manual” (2001, Cold Spring Harbour LaboratoryPress; ISBN: 0879695773); Ausubel et al., in:

Current Protocols in Molecular Biology (J. Wiley and Sons Inc, NY, 2003,ISBN: 047150338X); C. Dieffenbach & G. Dveksler: “PCR primers: alaboratory manual” (CSHL Press, ISBN 0879696540); and “PCR protocols”,by: J. Bartlett and D. Stirling (Humana press, ISBN: 0896036421).

Conveniently, the PCR reaction using the primers SEQ ID NO's: 30 and 31is combined with, and directly follows the RT reaction required toproduce cDNA; making the combined assay an RT-PCR assay, as described inthe examples.

An RT-PCR assay as described, has been applied by the inventors on avariety of samples; a positive results was obtained with samples of theisolates of the novel avian Astrovirus according to the invention; a“positive” PCR result being the visualisation of an approximately 260basepair reaction product after electrophoresis, in relation toappropriate positive and negative control samples. However, RT-PCRsamples prepared from the related avian Astroviruses ANV1 and ChickenAstrovirus 2 (CAstV2) were consistently found to be negative in the PCRusing the primers of SEQ ID NO's: 30 and 31.

An example of such a PCR test-result is represented in FIG. 5, showing aphotograph of an Ethidium bromide stained, UV-light illuminated, agarosegel, on which PCR products were run of a PCR using primers SEQ ID NO's:30 and 31. Only a sample of Isolate 19 reacted positive, samplescontaining ANV1 or CAstV2 were negative, as were negative controls.

Surprisingly, a highly remarkable and striking pathologic effect wasobserved upon infection and incubation of embryonated avian eggs with anavian Astrovirus according to the invention: affected embryo's displayeda bright-red staining of the legs and wing-tips. This has never beenobserved or described before. In addition, the infected embryo'sdisplayed other, but less distinctive, features: a generalised light redstaining, oedema of the abdomen, and a liver that was swollenconsideraby and had turned to a dark red/purple.

This specific pathology was observed about 2-7 days after infection ofembryonated SPF chicken eggs, and almost always led to embryo mortality,depending on the virus titre that was inoculated.

For comparison, inoculation of ANV1 into embryonated eggs was donealongside, using similar test conditions. ANV1 did also induce swellingof the liver, but in that case the livers were even darker in color, andthe overall color of the embryo was dark red. However, ANV1 neverinduced the specific bright red coloration of the legs and wing-tipsthat was seen for the isolates of the avian Astrovirus according to theinvention.

Therefore, in a further preferred embodiment, the avian Astrovirusaccording to the invention is characterised in that it is capable ofinducing a red coloration of the legs and wing-tips of chicken embryos,upon inoculation into embryonated chicken SPF eggs of about 5-7 daysold, followed by incubation for 2-7 days, or even longer whenappropriate.

As described, embryonated SPF chicken eggs and equipment for theirincubation are available commercially. Typically, incubation ofembryonated chicken eggs is done at about 35-39° C., in humidifiedatmosphere incubators.

Inoculation may be by any convenient route, e.g. the CAM, yolk sac orallantoic route.

As the skilled person will appreciate, the specific bright redcoloration of legs and wing-tips may fail to become visible inincidental cases where e.g. the amount of virus that was inoculated isextremely low (not allowing “take” of the virus) or extremely high(causing mortality before coloration could occur); or where the embryo'sare not of sufficient quality to allow viral replication. In general thespecific bright red coloration can be observed upon inoculation ofbetween about 10 and about 10̂6 EID50/egg (EID50=egg infectious dose 50%)

One particular isolate of the avian Astroviruses according to theinvention, named isolate 19, was further purified and amplified to serveas reference sample and be deposited. The isolate was purified bylimiting dilution on chicken embryo liver (CEL) cells. Each time thehighest dilution still inducing cpe on CEL cells was passaged on. Nextthis purified virus was amplified by three rounds of viral amplificationin the allantoic cavity of embryonated SPF chicken eggs. Allantoic fluidwas harvested and stored frozen. This purified virus was titrated onembryonated chicken SPF eggs, and was found to have a titer of 6.1 Log10EID50/ml. This was material was freeze dried in a standard stabiliserbuffer and stored at −20° C. Sterility was confirmed. Samples of thisvirus material were then deposited at the “Collection Nationale deCultures de Microorganismes” (CNCM) of the Institut Pasteur, in Paris,France, under deposit number: CNCM 1-3895, on 23 Jan. 2008.

Therefore, in a further preferred embodiment, the avian Astrovirusaccording to the invention is a virus as deposited under number CNCM1-3895 at the Collection Nationale de Cultures de Microorganismes(CNCM), of the Institut Pasteur in Paris, France.

Although the sample that was purified, amplified, and deposited was theisolate named “isolate19”, however, either one of the isolates of theavian Astrovirus according to the invention, described in Table 1, canserve as reference sample for the novel group of avian Astrovirusesaccording to the invention.

For in vitro culturing and for testing of the avian Astrovirusesaccording to the invention, several culture systems can be used: e.g.(avian) cells lines, such as primary cells like CEL or chicken embryokidney (CEK) cells, can advantageously be used. The preparation of CELor CEK cell-cultures from embryonated chicken SPF eggs is well known toa person skillled in the art.

Especially CEK cells are well suited to monitor viral growth, andmeasure viral amount and viability. Even though the cytopathic effect(cpe) that the avian Astrovirus according to the invention produces onthese cells is a-typical, infected cells can be clearly seen as roundedcells, curling up from the monolayer, and progression towardscell-lysis.

Therefore viral titration assays can be devised using progressivedilutions of viral samples, and scoring of the highest virus dilutionstill producing cpe. Viral titre can than be expressed as tissue cultureinfective dose 50% (TCID50) per mililiter as calculated by theSpearman-Kärber method (described in: D. Finney: Statistical method inbiological assay, C. Griffin & Co., Londen, ISBN 0195205677). Suchtechniques are all well known to a skilled person.

This way the virus can be cultured in tissue culture flasks ormicrotitration plates for a variety of tests and purposes. Commonly itis beneficial to first adapt viruses isolated from the field to in vitroculture on cells, by applying a few passages on such cells, before usein virus tests or titrations.

Alternatively, tests and titrations can be performed in embryonatedeggs, preferably chicken SPF eggs. For instance virus titration can bedone by inoculation of increasingly diluted virus samples into the yolksac of 6 day old embryonated SPF chicken eggs. After scoring the numberof infected or dead embryos, the resulting titer of the sample testedcan be expressed in El D50 per mililiter, for instance using theSpearman-Kärber method (supra).

For the deposited virus its capacity to cause pathology for hatchedchickens and turkeys was confirmed under laboratory conditions in anumber of experimental test chickens. Inoculation was via multipleroutes: intramuscular, oral, and ocular. Several of the inoculatedchickens developed severe signs of disease, whereas mock infectedchickens were without symptoms. Symptoms observed were mortality,general depression, and mild to severe diarrhoea. Upon histopathology,nephritis and enteritis were observed. No disease of the legs or jointswere observed, however that could also not expected as the chickens usedfor these experiments were too young (3-6 weeks), and/or not heavyenough (SPF chickens of layer type) to manifest such problems.

Of the deposited virus, the nucleotide sequence of additional regions ofthe genome is presented herein for further characterisation: ORF lb andORF 2. The respective nucleotide and putative amino acid sequences arepresented herein as SEQ ID NO's: 5-8, see Table 1.

The different primers that were used to determine these additionalnucleotide sequences are presented herein as SEQ ID NO's: 32-34, and aredescribed in Table 4; for convenience this table also includes the otherPCR- and sequencing primers described herein.

TABLE 4 Description of PCR-and sequencing primers as described herein  Location of   first base SEQ Inventors' on SEQ ID ID primer HybridisesNO: 1; Sequence NO: codes to: direction (5′→ 3′) RT reaction primer 3 173′ end-region 6731, rev TCg WTS CTA CYC of AAstV Detection primers 30 29Orf 1a of novel  2252, fwd gTY CTY ACC gAR gAR gAR TAY C 31 30 AAstV2499, rev AAD gTT ATY CTC CTA RgB TKH C according to the inventionSequencing primers 28 F-II Orf 1a of 2171, fwdAAA ggK AAg ACD AAg ARR RAC Mg 29 R-II-3 AAstV 2640, revTCg CCT TCT ggA Agg TCT TCA 32 20 Orf 1b of 3721, fwdTgg HCM CCY TTY TTY ggH g 33 21 AAstV 4020, revRTT RTC MAC DgT KgT DgA RWA YTg 34 ORF2-R Orf 2 of AAstV 6622, revTTA gAT CTg AAA gCg CCg gAg g AAstV = avian Astrovirus; rev = reverse;fwd = forward

A further method to provide additional characterisation of the avianAstroviruses according to the invention, with the deposited virus sampleas reference, is by determining its immunological features byserotyping, preferably by using VN- or IFT-assays.

In all tests, the novel avian Astrovirus proved to be immunologicallydisctinct from other avian viruses, as it could not be neutralised, andwas not specifically bound by, antibodies specific for a variety ofother avian viruses: Reovirus, Fowl Adenovirus (types 1, 2, 4, 5, and8), Infectious Bronchitis virus, Newcastle Disease virus, InfectiousBursal Disease virus, avian Enterovirus (isolates 1821/9 and FP3), Eggdrop syndrome and avian Influenza virus. This was also the case forantibodies specific for the avian Astroviruses ANV1 or CAstV2, whereasthese sera did effectively neutralise their donor viruses: ANV1 andCAstV2 up to a 1:640 or a 1:10240 dilution respectively.

However, a sample of the deposited isolate 19 virus was effectively, andselectively neutralised by a chicken antiserum generated against isolate19 in SPF chickens that were experimentally infected with the isolate 19virus. After giving booster inoculations, a specific antiserum could beobtained. Read-out used for the virus neutralisation in this assay wasthe prevention of embryopathology by the neutralisation.

The results of the immunologic experiments wherein specific antibodiesonly recognised and neutralised the avian Astroviruses according to theinvention, demonstrates that these viruses belong to a new and uniqueviral serotype.

This can be put to practice for instance in a further preferredembodiment, wherein the avian Astrovirus according to the invention ischaracterised in that said avian Astrovirus can be neutralised in avirus neutralisation assay, by antibodies directed against a sample ofthe Astrovirus as deposited under number CNCM 1-3895 at the CollectionNationale de Cultures de Microorganismes (CNCM), of the Institut Pasteurin Paris, France.

An alternative approach to the generation of antibodies using thedeposited virus according to the invention is also possible. Forexample, a virus-sample that is to be investigated for being (derivedof) an avian Astrovirus according to the invention might itself be usedto induce antibodies, which antibodies in turn can than be tested fortheir capacity to bind, or even to neutralise, an avian Astrovirusaccording to the invention. Conveniently such antibodies can be testedon a sample of the deposited virus according to the invention.

To this purpose, the test virus or a fraction or preparation thereof canbe inoculated into an animal. The animal used for generation of suchantibodies can in principle be any animal, but preferably a mouse, rat,rabbit, hamster, goat or chicken is used. Antibodies produced this waycan be tested for binding or neutralisation to a avian Astrovirusaccording to the invention, e.g. conveniently by ordering a sample ofthe deposited virus according to the invention from the CNCM. This canbe cultured, for instance on eggs, CEK or CEL cells, or othersubstrates, and finally incubated with the antibody raised with the testvirus sample. Detection of antibody binding can be done e.g. by IFT orVN assay.

This thus constitutes a method for the identification andcharacterisation of a live or inactivated virus, or of a fraction orpreparation thereof, as being or being derived from, an avian Astrovirusaccording to the invention.

Therefore, in a further preferred embodiment, the invention provides anavian Astrovirus according to the invention, which is characterised inthat said virus is capable of inducing antibodies that can neutralise asample of the deposited Astrovirus according to the invention in a VNassay.

Protocols for setting up a VN assays are well known in the art, and arewithin the routine capacity of a person skilled in the art. Preferencesand details for VN assays are described above.

As is evident from the results of the immunologic assays usingantibodies specific for the avian Astrovirus according to the invention,the invention also provides an antibody that can bind and that canneutralise an avian Astrovirus according to the invention.

Therefore, an other aspect of the invention is an antibody or fragmentthereof that can neutralise the avian Astrovirus according to theinvention, in a virus neutralisation assay.

Such antibodies according to the invention can be obtained and producedin a variety of ways, all wel known and available to the skilled person.For instance the antibodies can be produced in experimental animals by(repeated) inoculation as described above.

Alternatively antibodies can be produced by immortalised B-lymphocytecells, as in the hybrydoma technology. Modern improvements to theclassic hybridoma technology have made this technique more efficient andproductive, for instance by enhancing the amount of desired spleen cellsbefore fusion, by positive selection (panning), followed by expansion inculture with a mixture of cytokines. Also the fusion-technique has beenimproved, by changing from the classic poly-ethylene glycol fusion, toelectro-fusion.

In a further alternative approach, antibodies can be obtained throughthe use of molecular biological techniques and expression in recombinantexpression systems. Molecular biological techniques allow the adaptationof such antibodies to better match the signature of antibodies from thetarget species that is to be treated, or the design of antibodiescarrying two binding regions with different specificities. Expressionsystem development allows the set up of high volume expression systems,such as expression in plants.

Recombinant expression systems are also well known in the art and forinstance use: bacterial (e.g. Escherichia coli, Salmonella, Bacillus,Caulobacter etc.), yeast (e.g. Saccharomyces), insect (Spodoptera,Trichoplusia), mammalian (e.g. chinese hamster ovary), or plant(tobacco, potato, etc.) cells. These cells can be transformed with anexpression construct carrying the heterologous sequence to be expressed.Also, combined systems are known, using a recombinant micro-organismcomprising the nucleic acid to be expressed, which micro-organism can becultured on cells in vitro to produce the desired protein at the desiredscale. One example is the baculovirus/insect cell expression system.Finally, so-called cell-free expression systems are availablecommercially for cell-free expression of an appropriate recombinant DNAmolecule.

The term “an antibody or fragment thereof” is meant to indicate thatboth complete immunoglobulin molecules or parts thereof are consideredto be within the scope of the antibody according to the invention.Fragments of antibodies according to the invention are protein moleculesthat can still bind to an epitope of an avian Astrovirus according tothe invention. Examples are FAB, scFv, Fv, dAb, or Fd fragments, allwell known in the art.

Such fragments may be obtained from intact antibodies by e.g. chemicalor enzymatic digestion. Alternatively such fragments can for example beobtained from a recombinant expression system, for example aphage-display system.

As is well known to a skilled person, the binding of an antibody orfragment thereof to a virus encompasses the recognition of epitopes onthe virus particle. Most often these epitopes are formed by linear orthree-dimensional conformations of molecules displayed on the viralparticle. As a result, preparations of a virus or fractions thereofcould be bound by antibodies or fragments thereof according to theinvention, as long as these preparations or fractions still contain andpresent the right epitopes in the right form. In the case of the avianAstrovirus according to the invention the viral protein most prominentlypresented to the immune system is the capsid protein encoded from ORF 2.

The skilled person will also appreciate that when the antibody accordingto he invention was produced via inoculation of an animal, the animalserum obtained is a polyclonal antiserum, meaning that the antiserumcontains a mixture of antibodies that can bind to a wide variety ofepitopes. In practice this provides multiple interactions, which allowthe efficient binding of preparations or fragments of the viral protein,even though these may not possess, or may not properly present, all ofthe epitopes that are normally available on an intact live virusparticle.

Alternatively, the antibody according to the invention can be amonoclonal antibody, for instance as produced by the hybridomatechnology, or the antibody according to the invention can be anantibody fragment such as a Fab fragment. In this case the antibody orfragment has a much reduced capacity to recognize different epitopes, asit will normally only bind specifically to a single epitope.Consequently when this particular epitope is not present or notdisplayed on the virus fragment or preparation, it may not be bound atall by such a monoclonal antibody or antibody fragment.

Antibodies according to the invention can be used in a variety of ways;for characterisation of the avian Astrovirus according to the invention,for diagnostics, for therapy, and/or for quality assurance purposes.

The invention provides the production of the avian Astrovirus accordingto the invention on an industrial scale. This can be achieved byculturing said virus on host cells in in vivo or in vitro systems. Invitro systems comprise primary or immortalised cell-lines. Examples ofprimary cells are CEK or CEL cells. An example of an immortalisedcell-line is the LMH cell line (Kawaguchi et al., Cancer Res., vol. 47,p. 4460-4464). Examples of in vivo systems are culture in embryonatedavian eggs, or in inoculated birds. Especially cultures on cells or eggscan be scaled up conveniently; e.g. cell cultures can be kept incontainers of various sizes, such as flasks, roller bottles, or evenfermentors. Techniques and equipment for cell-culture technology at anyscale is well known and readily available from commmercial suppliers.

Therefore the invention provides in a further aspect a host cellinfected with an avian Astrovirus according to the invention.

Also, the invention provides a method of producing an avian Astrovirusaccording to the invention comprising multiplication of the avianAstrovirus according to the invention in an appropriate system, andharvesting the virus material.

The characterisations described herein of the avian Astrovirus accordingto the invention do not only refer to such avian Astroviruses in anintact, replication competent form. As a skilled person will appreciate,the present invention provides preparations of this virus in manydifferent forms. These comprise for instance preparations wherein theavian Astrovirus according to the invention has been inactivated, orpreparations wherein such a virus has been fractionated in one or moreways, (bio-)chemically or physically, for instance by extraction,lysation, digestion, homogenisation, or sonication.

Methods for inactivating virus are well known and for instance includechemical or physical inactivation, such as inactivation with formalin,beta-ethanolimine, or beta-propiolactone; also heating or irradiationwith UV-light, X-rays, or radioactive radiation.

Methods for fractionation also are known in multitude, for instanceextraction or lysation with a detergent such as Triton® X-100; digestionwith trypsin; homogenisation by freeze-thawing or French cell-press; orsonication by ultra-sound.

The starting material for such preparations can be a purified (live orinactivated) avian Astrovirus according to the invention, but also ahost cell according to the invention, or a cell-culture or part thereofcomprising a host cell or avian astrovirus according to the invention.For example such a part of a cell-culture may be a supernatant or apellet of a centrifugated cell-culture. Evidently these cell-cultures orparts thereof may themselves be turned into preparations such as anextract, lysate, digest, homogenate, or sonicate as described.

Such preparations of the avian Astrovirus according to the inventioncontain one or more antigens and epitopes of the avian Astrovirusaccording to the invention.

Therefore in a further aspect, the invention relates to an antigenicpreparation obtainable from the avian Astrovirus according to theinvention.

The antigenic preparation according to the invention can be bound byantibodies or parts thereof according to the invention, or can itself beused to induce binding or neutralising antibodies or parts thereof. Thiscan advantageously be applied in vaccines and diagnostics as will bedescribed below.

Antigenic preparations according to the invention, are for exampleproteins from the avian Astrovirus according to the invention, or partsof such proteins. These proteins or parts thereof can be obtained byisolation from such a virus. However, these proteins or parts thereofcan also conveniently be produced by recombinant DNA expressiontechniques using nucleic acids from the avian Astrovirus according tothe invention. For instance nucleic acid sequences of part of the ORF 1aregions as described in any one of SEQ ID NO: 4, 10, 12, 14, 16, 18, 20,22, 24 or 26 can be used; as described these cDNA sequences are relatedin that they have nucleotide sequence identity of at least 88% with SEQID NO: 4.

Therefore the invention provides in a further aspect a DNA moleculecomprising a region having a nucleotide sequence identity of at least88% with SEQ ID NO: 4.

Also nucleic acids of ORF 1b or ORF 2 can be used as described in SEQ IDNO: 6 or 8.

Such nucleic acid sequences and cDNA fragments can be cloned andexpressed using recDNA techniques well known in the art, for instance bysubcloning into a recombinant DNA molecule for further manipulation. TherecDNA molecule can be a vector such as a bacterial plasmid. Preferablythe nucleic acid from the avian Astrovirus according to the invention isoperatively linked to an expression control sequence, such as apromoter, allowing its expression in an appropriate host cell orexpression system. Alternatively the nucleic acid, cDNA, or recombinantDNA molecule can be introduced into a live recombinant carriermicro-organisms such as a bacterium or virus.

In analogy, based on the putative amino acid sequences described in SEQID NO's: 5, 11, 13, 15, 17, 19, 21, 23, 25, and 27, and theirrelatedness, the invention provides in a further aspect a proteincomprising a region having an amino acid sequence identity of at least93% with SEQ ID NO: 5.

These embodiments have great practical and advantageous utility in theapplication of vaccines and diagnostics as will be described below.

A vaccine can be prepared from live or inactivated microbiologicalpathogens, such as viruses, or from fragments of such micro-organisms.Such micro-organisms are commonly produced industrially in smaller orlarger volumes, by incubation on cells, organs or tissues, embryonatedeggs, or in host animals. After harvesting a suspension comprising themicro-organism, this suspension is formulated into a vaccine and thefinal product is packaged. After extensive testing for quality, quantityand sterility such vaccine products are released for sale.

The general techniques and considerations in vaccinology are well knownin the art and are described for instance in governmental regulationsand in handbooks such as: “Veterinary vaccinology” (P. Pastoret et al.ed., 1997, Elsevier, Amsterdam, ISBN: 0444819681), and: “Remington: thescience and practice of pharmacy” (2000, Lippincot, USA, ISBN:683306472).

A vaccine is commonly known in the art to represent a pharmaceuticalcomposition comprising an immunogenic compound in a pharmaceuticallyacceptable carrier. The immunogenic compound is a live or inactivatedmicro-organisms, or a subunit thereof, capable of inducing an activationof a targets' immune system.

Surprisingly it was found that the avian Astrovirus according to theinvention, and antigenic preparations, host cells, antibodies, nucleicacids, proteins, as well as methods for their preparation, culture,identification and quantification can be applied in vaccines and fordiagnostic purposes. Such vaccines serve to protect a target animal frominfection with the avian Astrovirus, or reduce the replication of saidvirus or the symptoms of the disease it causes. Such diagnostic assaysallow the detection of the virus or its antigens e.g. in animal fieldsamples or viral preparations.

Combined the vaccines and diagnostics allow the identification anddefence against infection and/or disease by the avian Astrovirusaccording to the invention.

Vaccine capacity was demonstrated by the animal experiments described,in which chickens were inoculated with the avian Astrovirus according tothe invention. This led to the production of highly specific antibodies.Moreover these antibodies were demonstrated to be able to neutralisespecifically the avian Astrovirus according to the invention. As is wellknown in the art, the induction of neutralising antibodies is animportant step in the feasibility of an effective and immuno-protectivevaccine.

The repeated inoculations of the test animals not only provided abooster to the antibody levels produced, but also served as challengeinfections, which could be tolerated and overcome by a large number ofthe birds in the test.

The avian Astrovirus according to the invention—or parts thereof—canthus be formulated in an appropriate pharmaceutical composition, and beapplied to avians as a vaccine.

It is within reach of a skilled person to further optimise this vaccine,for instance by fine-tuning the efficacy and safety of the vaccine, sothat it provides sufficient protection at an acceptable level ofvaccination reaction. This can be done by adapting the vaccine dose, orby using the a vaccine in another form or formulation, or by adaptingthe other constituents of the vaccine (e.g. the stabiliser or theadjuvant), or by application via a different route.

Well known variants of a vaccine for the invention for instance may usethe novel avian Astrovirus in a live or an inactivated form; may be asubunit vaccine when using the antigenic preparation and/or the proteinaccording to the invention; may be in the form of a passive vaccine whenusing the antibody or fragment thereof according to the invention; ormay be in the form of a DNA vaccine when using the DNA moleculeaccording to the invention.

Therefore another aspect of the invention provides a vaccine comprisingthe avian Astrovirus, the antibody or fragment thereof, the antigenicpreparation, the DNA molecule, or the protein all according to theinvention, and a pharmaceutically acceptable carrier.

In analogy, the invention relates in further aspects to a compound orcomposition for use in a vaccine for poultry, wherein the compound orcomposition is the avian Astrovirus, the antibody or fragment thereof,the antigenic preparation, the DNA molecule, or the protein allaccording to the invention.

In a further aspect the invention relates to the use of a compound orcomposition for the manufacture of a vaccine for poultry, wherein thecompound or composition is the avian Astrovirus, the antibody orfragment thereof, the antigenic preparation, the DNA molecule, or theprotein all according to the invention.

In a further aspect the invention relates to a method for themanufacture of a vaccine for poultry, wherein a compound or compositionis admixed with an appropriate pharmaceutical carrier, and wherein thecompound or composition is the avian Astrovirus, the antibody orfragment thereof, the antigenic preparation, the DNA molecule, or theprotein all according to the invention.

A “pharmaceutically acceptable carrier” can e.g. be sterile water, aphysiological salt solution, or a buffer suitable for the purpose. In amore complex form the carrier may itself comprise other compounds, suchas an adjuvant, an additional antigen, a cytokine, etc.

The vaccine according to the invention can be used both for prophylacticand for therapeutic treatment.

The term “vaccine” implies the presence of an immunologically effectiveamount of the avian Astrovirus according to the invention, and thepresence of a pharmaceutically acceptable carrier.

What constitutes an “immunologically effective amount” for the vaccineaccording to the invention is dependent on the desired effect and oncharacteristics of the avian Astrovirus and the target organism.Determination of the effective amount is well within the skills of theroutine practitioner, for instance by monitoring the immunologicalresponse following vaccination, or after a challenge infection, e.g. bymonitoring the targets' clinical signs of disease, serologicalparameters, or by re-isolation of the pathogen, in comparison to theresponses seen in unvaccinated animals.

The use of very high amounts of the avian Astrovirus, the antigenicpreparation, the antibody or fragment thereof, the DNA molecule, or theprotein according to the invention in a vaccine although immunologicallyvery effective, will be less attractive for commercial reasons. Apreferred amount of any of these compounds or compositions according tothe invention, comprised in a vaccine according to the invention, isbetween 0.1 and 90% of the final volume of the vaccine. More preferablythe amount is between 1 and 50%, 1 and 25%, and 1 and 10%, in that orderof preference.

In case of an inactivated- or subunit vaccine, the compound orcomposition according to the invention can be expressed in ELISA units.Amounts of ELISA units that are effective need to be determined inrelation to the Elisa unit of a standardised sample giving a certainefficacy.

In case of a live vaccine according to the invention, an amount of pfu(plaque forming units), cfu (colony forming units), TCID50, EID50, orELD50 (embryo lethal dose 50%) can be used, depending on what is aconvenient way of quantifying the avian Astrovirus vaccine dose. Forinstance a live avian Astrovirus vaccine dose in a range between 1 and10̂6 EID50 per vaccine dose can advantageously be used; preferably arange between 10 and 10̂5 EID50/dose, more preferably between 10̂2 and 10̂4EID50/dose.

In a preferred embodiment the invention relates to the vaccine accordingto the invention wherein the avian Astrovirus according to the inventionis in live form.

Live vaccines in general have the advantageous properties that onlylittle inoculum is required, as the microorganism replicates itself.Also this generally induces a solid and effective immune response, withadequate immunological memory.

As known in the art, live vaccines can conveniently be applied asattenuated live vaccines, meaning the vaccine virus has a reducedvirulence or pathogenicity compared to the original viral isolate.Methods for viral attenuation are known in the art and comprise forinstance: continued passageing throug animals or over cell-cultures;chemical attenuation; or attenuation by molecular biological techniques.

In an alternate preferred embodiment the invention relates to thevaccine according to the invention wherein the avian Astrovirusaccording to the invention is inactivated.

Methods and materials for viral inactivation have been described above.Such inactivated vaccines in general have the advantage of being moresafe then live vaccines, as they do not expose the host to anypotentially pathogenic replicating live avian Astrovirus.

In a further preferred embodiment, the vaccine according to theinvention additionally comprises an adjuvant.

An adjuvant is an immunostimulatory substance boosting the immuneresponse of the host in a non-specific manner. Many different adjuvantsare known in the art. Examples of adjuvants frequently used aremuramyldipeptides, lipopolysaccharides, several glucans or glycans,Carbopol®, Aluminum-hydroxide (Al(OH)₃). These can be combined withdifferent emulsions, such as water in oil (w/o) emulsions, o/w emulsionsand w/o/w double-emulsions. Oil adjuvants suitable for use in oilyemulsions are e.g. mineral oils or metabolisable oils. Mineral oils aree.g. Bayol®, Marcol® and Drakeol®; metabolisable oils are e.g. vegetableoils, such as peanut oil and soybean oil, or animal oils such as thefish oil squalene. Alternatively a vitamin E (tocopherol) solubilisateas described in EP 382,271 may advantageously be used.

Very suitable o/w emulsions are e.g. obtained starting from 5-50% w/wwater phase and 95-50% w/w oil adjuvant, more preferably 20-50% w/wwater phase and 80-50% w/w oil adjuvant are used.

The amount of adjuvant added depends on the nature of the adjuvantitself, and on information with respect to such amounts provided by themanufacturer.

Whereas it is common practice to use adjuvants in combination withinactivated vaccines, several live vaccines have shown to also benefitfrom inclusion of an adjuvant. Therefore this embodiment is also withinthe scope of the invention.

In a further preferred embodiment the vaccine according to the inventionadditionally comprises a stabiliser.

A stabilizer can be added to the vaccine according to the invention e.g.to protect it from degradation, to enhance the shelf-life, or to improvefreeze-drying efficiency. Useful stabilizers are i.a. SPGA (Bovarnik etal., 1950, J. Bacteriology, vol. 59, p. 509), skimmed milk, gelatine,bovine serum albumin, carbohydrates e.g. sorbitol, mannitol, trehalose,starch, sucrose, dextran or glucose, proteins such as albumin or caseinor degradation products thereof, buffers, such as alkali metalphosphates, and polyamines such as spermine.

Also preservatives may be added, such as thimerosal, merthiolate, orgentamicin.

In addition, the vaccine may comprise one or more suitablesurface-active compounds or emulsifiers, e.g. Span® or Tween®.

The vaccine may also comprise a so-called “vehicle”. A vehicle is acompound to which the polypeptide or the protein according to theinvention adheres, without being covalently bound to it. Such vehiclesare i.a. bio-microcapsules, micro-alginates, liposomes and macrosols,all known in the art. A special form of such a vehicle is an immunestimulatory complex (ISCOM).

It goes without saying that admixing other stabilizers, carriers,diluents, emulsions, and the like to vaccines according to the inventionare also within the scope of the invention. Such additives are describedin well-known handbooks such as: “Remington”, and “VeterinaryVaccinology” (both supra).

It is highly efficient to formulate the vaccine according to theinvention as a combination-vaccine, as in this way multiple immunologicagents can be administered at once, providing reduction of time- andlabour costs, as well as reduction of discomfort to the vaccinatedtarget animals.

Such combination vaccines are achieved by combining the vaccineaccording to the invention with another antigenic compound, such as a(live or inactivated) virus, bacterium, parasite, or parts thereof suchas a protein, a carbohydrate, or a nucleic acid capable of encoding anantigen. As the vaccine according to the invention is intended for aviantarget animals, it is advantageously combined with an additional antigenthat is, or is derived from, an other poultry pathogen.

Therefore, in a further preferred embodiment, the vaccine according tothe invention is comprising at least one additional antigen obtainablefrom a micro-organism pathogenic to poultry.

Many avian pathogens are known, however some of the more relevantveterinary-economic pathogens are:

-   viruses: infectious bronchitis virus, newcastle disease virus, avian    influenza, Adenovirus, Egg drop syndrome virus, Infectious bursal    disease virus (i.e. Gumborovirus), chicken aneamia virus, Avian    encephalo-myelitis virus, Fowl Pox virus, turkey rhinotracheitis    virus, Duck plague virus (Duck virus enteritis), Pigeon Pox virus,    Marek's Disease, Avian Leucosis virus, Infectious laryngotracheitis    virus, Avian pneumovirus, and Reovirus;-   bacteria: Escherichia coli, Salmonella spec., Ornitobacterium    rhinitracheale, Haemophilis paragallinarum, Pasteurella multocida,    Erysipelothrix rhusiopathiae, Erysipelas spec., Mycoplasma spec. and    Clostridium spec.;-   parasites: Eimeria;-   fungi: e.g. Aspergillus, etc.

Also conceivable is the combination in the vaccine according to theinvention of two or more avian Astroviruses according to the invention,for instance to form a combination vaccine that is more broadlyeffective against variants of the avian Astrovirus according to theinvention. Examples of avian Astroviruses that may be used to form thecombination vaccine are the isolates described in Table 1.

A vaccine according to the invention may take any form that is suitablefor administration to poultry, and that matches the desired route ofapplication and desired effect.

The preparation of a vaccine according to the invention is carried outby means well known to the skilled person. Preferably the vaccineaccording to the invention is formulated in a form suitable forinjection, such as a suspension, solution, dispersion, emulsion, and thelike. Commonly such vaccines are prepared sterile.

Many ways of administration can be applied, all known in the art. Thevaccines according to the invention when inactivated, are preferablyadministered by injection; live vaccines, are preferably administered bya method of mass application such as via the feed, spray or drinkingwater.

The protocol for the administration of the vaccine according to theinvention ideally is integrated into existing vaccination schedules ofother vaccines.

Preferred target for the vaccine according to the invention is an aviananimal. More preferred targets are selected from the group consisting ofchicken, turkey, duck and goose. Most preferred target is chicken.

The dosing scheme for applying the vaccine according to the invention toa target organism can be in single or multiple doses, which may be givenat the same time or sequentially, in a manner compatible with theformulation of the vaccine, and in such an amount as will beimmunologically effective.

The age, weight, sex, immunological status, and other parameters of theavian target to be vaccinated is not critical, although it is evidentlyfavourable to vaccinate healthy targets, and vaccinate as early aspossible to prevent a field infection. For instance poultry cantherefore be vaccinated at the day of hatching, or even earlier, whilestill in ovo at 3-4 days prior to hatch; for example at 18 days ofembryonic development (ED) for chickens or at 24 days ED for turkeys.

For reasons of stability or economy a vaccine according to the inventionmay be freeze-dried. In general this will enable prolonged storage attemperatures above zero ° C., e.g. at 4° C. Procedures for freeze-dryingare known to persons skilled in the art, and equipment for freeze-dryingat different scales is available commercially.

Therefore, in a preferred embodiment, the vaccine according to theinvention is in a freeze-dried form.

To reconstitute a freeze-dried vaccine composition, it is suspended in aphysiologically acceptable diluent. Such a diluent can e.g. be as simpleas sterile water, or a physiological salt solution such as (phosphatebuffered) saline, or the diluent may already contain an adjuvatingcompound, such as a tochopherol, as described in EP 382,271. In a morecomplex form the freeze-dried vaccine may be suspended in an emulsione.g. as described in EP 1,140,152.

Therefore in a further preferred embodiment, the invention relates to avaccine composition obtainable by reconstitution of a freeze driedvaccine according to the invention.

In addition to the various uses of the comounds and compositionsaccording to the invention in vaccines, these can also be advantageouslyapplied in diagnostics, to detect the virus according to the inventionor its antigens or nucleic acids e.g. in animal field samples or viralpreparations.

This is also demonstrated by the results of the serotyping experimentsby IFT and VN assay as described above, wherein antisera against thedeposited avian Astrovirus according to the invention were applied todiscriminate between viruses falling within the scope of the invention(the isolates as described in Tablel) and those that did not (amongstmany others: ANV1, CAstV2, Reovirus etc.). Equally, the PCR experimentsdescribed demonstrated the capability to selectively identify the avianAstrovirus according to the invention.

As the skilled person will appreciate, this not only applies to theantibodies according to the invention, but also for the other compoundsand compositions according to the invention. Such diagnostic assays forinstance use the antibodies or fragments thereof according to theinvention to test for antigen; or use antigen (the virus, the antigenicpreparation, or the protein according to the invention) to test forantibodies thereagainst; or use nucleic acid (RNA from the virus, or theDNA molecule according to the invention) to set up hybridisation or PCRassays.

Therefore, in a further aspect, the invention relates to a diagnostickit comprising the avian Astrovirus, the antigenic preparation, theantibody or fragment thereof, the DNA molecule, or the protein, allaccording to the invention.

The term “diagnostic kit” implies features relating to a package forcommercial sale, for instance comprising a number of components anddisposables for performing a diagnostic test and an instruction leaflet;all in a commmercially feasible package form.

Such a diagnostic kit e.g. thus comprises the materials required for anantibody ELISA. In such a test for instance the wells of an ELISA platehave been coated with the avian Astrovirus according to the invention,to form an antibody ELISA kit, for the detection of antibodies againstthe avian Astrovirus according to the invention or preparations orproteins thereof. After incubation with the test-sample, labelledantibodies reactive with the immunoglobulins of the test sample (ifpresent) are added to the wells. A colour reaction then reveals thepresence in the test sample of specific antibodies.

Similarly, an antigen ELISA kit can be devised, comprising e.g.microtitration plates coated with the virus, the antigenic preparation,or the protein according to the invention.

The design of such immunoassays may vary. For example, the immunoassaymay be based upon competition, in stead of on direct binding.Furthermore, such tests may also use particulate or cellular material,in stead of the solid support of a device. The detection of theantibody-antigen complex formed in the test may involve the use oflabelled antibodies, wherein the labels may be, for example, enzymes orfluorescent-, chemo luminescent-, radio-active- or dye molecules.

Advantageously, the vaccines and diagnostics according to the inventioncan now be used in cooperation to devise an approach for the eradicationof the avian Astrovirus according to the invention in a certain regionor animal population.

LEGEND TO THE FIGURES

FIG. 1: displays a multiple alignment of the cDNA sequences of a sectionof ORF 1a from a selected number of isolates of the avian Astrovirusesaccording to the invention. Reference is the sequence from Isolate 19.Outside reference is the corresponding part of the cDNA sequence of thegenome of an ANV1 reference virus (SEQ ID NO: 1). The boxed area marksthe 12 nucleotide region, which is not present in the ANV1 ORF 1a.

FIG. 2: displays a multiple alignment of the putative amino acidsequences, as prepared by computer translation of the cDNA sequenceslisted in FIG. 1. The boxed area marks the 4 amino acids, that are notpresent in the protein encoded by ORF 1a of ANV1.

FIG. 3: displays a dendrographic tree of the results of the nucleotidesequence alignments as presented in FIG. 1.

FIG. 4: presents a graphic representation of the hybridisation areas ofprimers SEQ ID NO's: 30 and 31, in relation to the location of the 12nucleotide region (represented in bold) which is unique for the avianAstroviruses of the invention.

FIG. 5: shows a photograph of an Ethidium bromide stained, UV-lightilluminated, agarose gel, on which the electrophoresis was run of theproducts of a PCR assay using primers SEQ ID NO's: 30 and 31 on a numberof cDNA samples. The expected product of a positive PCR, a band ofapproximately 260 basepairs is clearly visible, in lane 5 only. Thepreceeding RT reactions had been done with the primer of SEQ ID NO: 3.

Lane 1: Negative control (no cDNA in PCR reaction)

Lane 2: Liverhomogenate from uninfected chicken

Lane 3: ANV1; a German isolate of 1991, amplified 1× CAM, 4× yolksac,allantoic fluid harvested.

Lane 4: CAstV2; derived from the virus as deposited at the CNCM undernumber CNCM 1-2932, allantoic fluid.

Lane 5: Isolate 19 of the avian Astrovirus according to the invention,allantoic fluid

M: DNA size marker lane: bands at 200, 400, 600, 800, 1000 bp, etc.

The invention will now be further described with reference to thefollowing, non-limiting, examples.

EXAMPLES Example 1 Isolation of an Avian Astrovirus from Field Samples

Many isolates of avian Astrovirus according to the invention have beenisolated from chickens and turkeys, from various farms in theNetherlands and Germany. As a typical example, the isolation of samplenr. 161319 (“Isolate 19”) is described here in detail.

Isolate 19 Astrovirus was isolated from a pool of tracheas from 4 layerhens of 36 weeks old, on a Dutch farm, presenting a drop in feedconsumption, drop in egg production and increase of second grade eggs.Also generally suffering from indigestion, diarrhea, and mobilityproblems.

Pooled tissue (0.5-1.0 g) of tracheas of the 4 birds were mixed withabout 10 ml of “rinsing-medium” (containing: 500 ml HBSS [Hank's basicsalt solution]+2 ml Benzyl Penicilline Natrium (1.000.000 I.E/ml)+8 mlStreptomycine (250 mg/ml)+0.5 ml Fungizone (2.000 μg/ml)+2 ml of NaHCO₃7.5%) and homogenised using a stomacher during 2 minutes. The suspensionwas centrifugated during 15 minutes at 2000×g at 2-8° C. The supernatantwas then filtrated using a 450 nm filter.

The resulting fluid was inoculated into the allantoic cavity of four 8-9day-embryonated chicken SPF eggs (1 ml per egg), the yolk of four 5-6day-embryonated SPF eggs (1 ml per egg), and the CAM of four 9day-embryonated SPF eggs (0.5 ml per egg) and incubated at 36.5 to 38.5°C. in 40 to 55% humidity. The eggs were candled daily. Embryonic deathoccurring within one day of inoculation was considered to benon-specific.

The embryos of the yolk-sac inoculated eggs did not show anyabnormalities at 7 days p.i. Yolk fluid was harvested nonetheless andused for a second passage.

Four ml of the harvested yolk fluid from the first passage (1 ml peregg) was mixed with 0.5 ml of “passage medium” (containing: HBSS 90 ml+2ml Benzyl Penicilline Natrium (1.000.000 I.E/ml)+8 ml Streptomycine (250mg/ml)+0.5 ml Fungizone (2.000 μg/ml)+2 ml of NaHCO₃ 7.5%). From thismixture 1 ml per egg was inoculated in the yolk of five 5day-embryonated SPF eggs and incubated at 36.5 to 38.5° C. with 40 to55% humidity. The eggs were candled daily.

On the sixth day after inoculation, one embryo had died and showedredness of the body. The allantoic fluid from this egg was tested forReovirus in agar gel precipitation (AGP) assay; it was negative forReovirus.

About 4 to 5 ml of yolk of this egg with the abnormal embryo washarvested and used for further passages, in the same way as the secondpassage.

In the third passage, on the fifth day after inoculation, three embryoshad died and two of them showed redness of the body. The pooledallantoic fluid was negative in the AGP for Reovirus, and for Adenovirustype1.

In further passages the titer of the isolate increased, as embryo's diedor showed symptoms earlier after inoculation. In the 6^(th) and 7^(th)passage the typical bright red staining of the legs and wingtips becameapparent.

Consistently, allantoic fluid was negative in HA or AGP tests for IBDV,Reovirus, Adenovirus, NDV, AIV and IBV. Also a PCR assay for IBDV wasnegative.

Harvested CAM and allantoic fluids were stored frozen at −80° C., andused for further characterisation.

Example 2 Viral RNA Isolation

For isolation of the total viral RNA, a QIAamp® Viral RNA mini Kit(QiaGen™) was used, according to the manufacturer's instructions. Inshort: 140 μl virus-containing fluid, such as allantoic fluid, or atissue- or embryo homogenate (typically in quantities between 5-50%w/v), was mixed with prior prepared extraction buffer and carrier RNA.This was incubated at room temperature for 10 minutes. Next pure ethanol(96-100%) was added, mixed, and the mixture was applied to QIAamp Minispin column (in a 2 ml collection tube). This was centrifuged for 1 min.at 6000×g. The flow-through was discarded. The column was washed withwash buffer containing pure ethanol. Finally, the column was eluted with60 μl elution buffer. Commonly the RNA isolation was followed directlyby production of cDNA via RT reaction.

Presence of Astroviral RNA was verified by standard agarose/formamideRNA gel-elektrophoresis.

Example 3 Production of cDNA via RT Reaction

First strand cDNA synthesis was done using Amersham-Pharmacia™Ready-To-Go® You-Prime First-Strand Beads, according to themanufacturer's instructions. In short: 29 μl of the eluted RNA from thetotal viral RNA isolation, was denatured for 10 minutes at 65° C., and 2minutes on ice. This was added to a tube containing two beads, and 4 μlof 10 μM primer 17 (SEQ ID NO: 3) was added, to an endvolume of 33 μl.This was incubated for 1 minute at room-temperature, mixed by carefullpipetting, and incubated for 1 hour at 37° C. cDNA was stored frozenbelow −20° C. until further use.

Example 4 PCR Reactions

PCR was used to prepare double stranded (ds) DNA from cDNA out of the RTreaction. Also PCR was used as a diagnostic type detection assay toidentify specifically if avian Astrovirus according to the invention waspresent in a sample.

PCR reaction ingredients were from HT Biotechnolgy™ Ltd.: Supertaq®buffer (10× conc.) used at 1× conc.; Supertaq® Plus polymerase enzyme (5U/μl) used at 1 U/μl, and preMixed dNTP's (10 mM), used at 2 mM. Thesewere mixed in distilled, RNAse-free water.

Primers used were those as listed in Table 4. Primers were synthesisedby Invitrogen™, the Netherlands, and the primer stock solution was madein TE buffer (pH 8.0) at 100 μM; the working-stock solution was at 10 μMdilution for direct use.

For a standard PCR, the following ingredients were used: 27 μl Aquadest., 5 μl 10× Supertaq Plus buffer; 1 μl (equals 1 Unit) Supertaq Plusenzyme; 5 μl 2 Mm dNTP mixture; 5 μl each of forward and of reverseprimer (at 10 μM); and finally 2 μl of cDNA from the RT reactionmixture; reaching a total volume of 50 μl.

Typical reaction conditions, for instance for amplifying ORF 1a withprimers F-II and R-II-3 (SEQ ID NO: 28, 29), or ORF 1b by using primers20 and 21 (SEQ ID NO: 32, 33) were:

-   1 min. at 95° C.,-   40 cycles of: 30 sec. at 94° C.; 1 min. at 48° C.; and 40 sec. at    72° C.,-   10 min. at 72° C.-   hold at 20° C.-   NB: all PCR temperatures are ±about 1° C.

During optimisations, some variations were applied: 45 instead of 40cycles, and variations to the annealing temperature and -time in thecycling step: temperatures between 46 and 53° C. were used, at durationsof 30 sec or 1 minute.

For the specific detection of the avian Astrovirus according to theinvention, using primers 29 and 30 (SEQ ID NO: 30, 31) the optimalconditions used for the cycling phase were: 45 cycles, with an annealingstep of 30 sec. at 51 ° C. Other conditions were as for the primers20/21 reaction.

The PCR reactions for the cycle sequencing reactions had an essentiallydifferent set-up: 25 cycles of: 10 sec. at 96° C.; 5 sec. at 50° C.; and2 min. at 60° C., see below.

Example 5 DNA Analysis by Aqarose Gel-Electrophoresis, Purification andSubcloning

Agarose gel-electrophoresis was used for detection and visualisation ofthe PCR products produced. Also this served to purify PCR products byexcision and extraction, allowing the sequencing, or subcloning of theseisolated PCR products.

In short, 0.8% Agarose (low electro-endosmosis type) gells were cast inTAE buffer (pH 8.0), containing about 50 μl of an 0.5 mg/ml solution ofEthidium-Bromide per 100 ml agarose solution. A sample of the PCRproduct was mixed 1:10 with standard sample loading buffer(glycerol/SDS//bromophenol blue). Typically a marker lane was includedusing SmartLadder® from Eurogentec™. Electrophoresis, submerged in TAEbuffer, was performed typically for 1 hour at 100 V for a 20×20×1 cmgel, or until the blue dye reached the end of the agar. Visualisationwas by UV light. Gels were photographed with a digital camera.

Agar gel electrophoresis was also used to estimate the DNA concentrationof an excised and purified band; in that case a sample was run alongsideto a number of lanes holding different quantities of the DNA markerladder.

The purification of specific bands of PCR products from an agarose gelwas done using the QIAquick® Gel Extraction kit (QiaGen™) according tothe manufacturer's instructions, using either a micro-centrifuge or theQIAvac® vacuum manifold.

DNA bands excised and purified from agarose gel were subcloned intobacterial plasmids for further use, such as cloning, sequencing,expression, or hybridisation-detection assays. DNA fragments were clonedinto plasmid pCR2.1 using a TOPO-TA® vector and TOPO-TA® cloning kit(Invitrogen™), according to the manufacturer's instructions

Example 6 DNA Sequencing

DNA sequencing was performed by standard PCR cycle-sequencing, using aBig Dye® Terminator Ready Reaction Mix (ABI Prism™), and an ABI Prism™310 automated sequencing apparatus, all according to the manufacturer'sinstructions.

Typically 20 to 70 ng of DNA (purified PCR product, or cloned fragmentin vector) were used in a sequencing reaction, with 8 μl Big Dye®Terminator Ready Reaction Mixture, 1 μl of 10 μM primer, in distilledwater to a total volume of 20 μl. The primers used for sequencing wereusually the same as those used in the PCR to prepare the ds DNA productfrom the cDNA.

Cycle sequencing PCR was by 25 cycles of: 10 sec. at 96° C., 5 sec. at50° C., and 2 min at 60° C.

After PCR, the samples were purified using DyeEx® Spin columns(QiaGen™), for removal of Dye-Terminator, according to themanufacturer's instructions, using Eppendorf® tubes and amicro-centrifuge. The final eluate was resuspended 1:2 to a total volumeof 40 μl with distilled water.

Sequence determination was then done by analysis on an ABI Prism® 310Genetic Analyzer, with Data Collection version 1.0.4 en SequenceAnalysis version 3 software.

Sequence analysis was done using a number of programs, most used wereSequencher® (Gene Codes™), and Clone Manager® (Sci. Ed. Software™).Start and end sequences were trimmed to remove nucleotide readings thatwere introduced based on the denatured PCR primers that were used.

The DNA sequences presented herein in SEQ ID NO's: 4-26 (even numbers),are the consensus sequences derived from multiple sequencing reactions.Most times the sequences were also read from both two strands, usingforward and reverse sequencing primers; only SEQ ID NO: 8 was determinedfrom only one strand. Average redundance was about 4× per nucleotide.

Example 7 Titration of Astrovirus on Embryonated Chicken Eggs

Serial ten-fold dilutions of Astrovirus suspension were inoculated intothe yolk sac of six-day-old embryonated SPF chicken eggs using a 23G 1″needle. Subsequently, the eggs were incubated for seven days at 37° C.in an egg incubator.

The eggs were candled daily. Mortality occurring within 24 hours postinoculation was considered non specific and therefore such eggscontaining early dead embryos were discarded and excluded from thecalculation of the infectivity titre.

Embryos dying from 2 till 7 days post inoculation were inspected for thepresence of lesions characteristic for infection with the Astrovirusaccording to the invention; this was the case if the dead embryosexhibited bright red legs and wing-tips, and a swollen dark-red liver.The infectivity titre was calculated using a computer program based onthe method of Spearman & Kärber and was expressed in ELD50 per ml.

Example 8 Production of Astrovirus on Embryonated Chicken Eggs

Nine-day-old embryonated SPF chicken eggs were inoculated with 10̂5 ELD50of Astrovirus per egg in the yolk sac using a 22G 1½″ needle.Subsequently, the eggs were incubated at 37° C. in an egg incubator.

After 24 hours of incubation the eggs were candled. Death occurringwithin 24 hours was considered non-specific, these eggs were removed.

After 48 hours of incubation the allantoic fluid of all remaining eggswas harvested. The allantoic fluid was stored frozen below −60° C.

The infectivity titre was established by titration in embryonatedchicken eggs or on cells as described above.

Example 9 Production of Primary Cells

CEL cells were prepared from 14-16 day old embryonised SPF chicken eggs,by isolating the liver from the embryo. The liver was washed withPBS/phenol red, and incubated for 8-10 minutes at room temperature in asolution containing trypsin in PBS. Supernatant is harvested through a100 μm gauze. This is repeated two more times, next cell are collectedby centrifugation and resuspended in a suitable rich culture mediumcontaining 5% fetal calf serum (FCS).

CEK cells were prepared in a similar way, using the kidneys from about18 days old SPF chicken embryos.The kidneys were gently stripped,washed, and trypsinised once, for 20 minutes at room temperature. Next,CEK cells were filtered and taken up in culture medium+FCS.

Example 10 IFT Assay

Immunofluorescence tests (IFTs) were used to investigate thecross-species specificity of certain anti-sera, as well as for virustitration. Commonly IFT was performed on primary cells in microtitrationplates.

When CEK cells were used for an IFT, these were seeded at 10̂5 cells/100μl in the wells of 96-well microtitration plates. The cells were in astandard rich culturing medium, with 2% FCS and antibiotics, andincubated at 37° C. in 5% CO₂ atmosphere. The next day, virus wasinoculated onto the cells.

For titration purposes, serial dilutions of virus were used. Plates werethen incubated for a further 2 days. Then, supernatant was removed, andthe cells were fixed with pure ethanol at −70° C. The plates could bestored at −20° C. until use. To visualise the virus for the titration,the plates were stained with specific antiserum. Therefore, the plateswere adapted to room temperature, the alcohol was poured off and theplates were washed with PBS. A dilution of anti-Astrovirus serum wasprepared at a strength which was known to give good fluorescence, butnot too much background. This first antiserum was then brought onto theplates and incubated for 1 hours at 37° C. in a moist atmosphere. Theplates were then washed 3× with PBS. Then the second antibody-conjugatewas prepared, a goat anti-chicken IgG-FITC conjugate (Nordic™). This wasbrought onto the plates in the required dilution, and was incubatedagain for 1 hour at 37° C. After this incubation the plates were againwashed 3× with PBS, after which a 1:1 mixture of PBS:glycerol was added.Plates were then stored in the dark at 4° C. until reading byfluorescence microscope. A positive signal is the detection of specificfluorescence, correlating to signs of cpe in the cell-layer.

For serum-characterisation, and species cross-reaction tests,micro-titration plates were prepared in a similar way, using CEK or CELcells. Next day these were infected with the different viruses to betested, e.g. Astrovirus isolates according to the invention, but alsoANV1, and CAstV2, as well as other avian pathogens: Reovirus, IBV, NDV,FPV, Adenovirus, AIV, etc. The amount of virus was a fixed amount or adilution whatever was convenient. The plates were incubated for 2-3days.

To test the capacity of a certain antiserum to bind to differentviruses, the serum was incubated on the plates, and signal was enhancedby incubation with conjugate. For optimisation ofbinding—versus—background signal, a number of dilutions of the antiserain PBS were tested on different dilutions of the various viruses.

Example 11 Infection of Chickens with Astrovirus

The avian Astrovirus according to the invention was tested in chickensin an experimental set-up to reproduce the disease symptoms observed inthe field, and to re-isolate the micro-organism from these secondairyinfected animals. This served to comply with Koch's postulates toestablish the isolate tested as a pathogenic and virulent micro-organismand causative infectious agent of the field-symptoms observed.

Also, chickens were repeatedly infected to test vaccination efficacy bya live inoculum, against subsequent challenge infections.

Finally, total serum was isolated from experimentally infected chickensto obtain specific polyclonal antiserum against the inoculatedAstrovirus.

To this purpose, SPF layer type chickens, of white leghorn breed andboth sexes, were transferred to a negative pressure isolator at threeweeks of age. Animals were assigned to treatment groups by randomselection, and were individually labelled by double wing-bands. Standardfood and drinking water were available ad libitum.

At regular intervals througout the experiment blood samples were taken,also at day zero, to determine immune status. Next 15 chickens wereinoculated by three routes: with 0.2 ml each by ocular and byintramuscular route, as well as with 0.5 ml by oral route, at 10̂5EID50/0.2 ml dose, and 10̂5.4 EID50/0.5 ml dose. The inoculum was theisolate 19 Astrovirus as deposited, resuspended in water for injection.5 chickens, placed in the same isolator were not inoculated to serve ascontrols, and sentinels. Animals were inspected daily for clinical signsof disease or mortality.

At day 20 post inoculation (p.i.), a number of chickens were bled andsubmited for post-mortum histo-pathological examination. At day 23 p.i.the other (previously inoculated) chickens were boosted with a similardose of inoculum. After another 23 days, the experiment was terminated,living animals were bled and examined.

Some birds were uninoculated initially but were housed in the sameisolators as the inoculated/infected birds; as expected these birdsbecame infected through horizontal spread of the Astrovirus.

Example 11a Histopathology Results

The histopathological results of the animal trials were as follows: 2chickens had died, and 5 animals, of which 3 controls had diarrhea. Uponhistopathology several animals demonstrated more or less severepathology to the kidney and intestinal tract already from day 7 p.i. Thekidneys presented a severe interstitial nephritis, and tubulardegeneration as most prominent signs. Thymus and bursa showedlymphocytolysis; the pancreas showed apoptosis and acinar atrophy; andthe duodenum showed blunted and fused villi.

The horizontal infection of the uninoculated control chickensdemonstrated the virulence and infectivity of the Astroviral inoculum.

Example 11b PCR Results

Kidney samples of all chickens were tested by PCR for signs of the avianAstrovirus according to the invention. After homogenisation, and RNAisolation, RT samples were made. These were tested by PCR with primersof SEQ ID NO: 30 and 31. All samples of infected animals testedpositive, by presenting the specific 260 by band identifying the avianAstrovirus according to the invention. Appropriate positive and negativecontrols were included.

This demonstrated that the causative agent of the mortality andhistopathological signs was the avian Astrovirus according to theinvention. The symptoms observed, nephritis and diarrhoea, concurredwith those seen in the field. Problems to the leggs could not bereproduced; most likely the birds used where of too slender build toexibit such problems, and/or the conditions of SPF chickens in anisolator did not sufficiently mimic the multiple infective pressure abird in the field experienced.

Example 11c VN Results

Fixed amounts of Astrovirus isolate 19 were incubated with chickenantisera from day 0, day 20 and day 46 p.i., for 1 hour at 37° C. Thesevirus samples were then inoculated into 6 day old embryonated SPF eggs,and incubated for several days. At 4 days after egg-inoculation,embryo's receiving virus treated with day 0 serum had died, and showedtypical signs of infection by the avian Astrovirus according to theinfection.

However, virus incubated with serum taken at day 20, or day 46 serum hadnot affected the embryos. This demonstrates that isolate 19 virus can beeffectively neutralised by a specific chicken antiserum.

Also this proves that effective antisera with VN capacity can be inducedin chickens upon live inoculation with the avian Astrovirus according tothe invention.

Example 11d IFT Results

Results of cross-reaction IFT assays using the chicken polyclonalantisera demonstrated that an anti-Isolate 19 antiserum did notrecognise any virus other than the virus isolates of the avianAstrovirus according to the invention.

Similarly, antisera specific for ANV1 or for CAstV2 equally onlyrecognised the specific virus against which they had been raised.Equally important was that neither the anti-ANV1 nor the anti-ChAstV2serum bound to a virus-isolate of the avian Astrovirus according to theinvention. This applied even to the highest antibody amounts tested (theleast diluted samples).

Also, none of the antisera against other avian pathogens, mostrelevantly Reovirus, could bind to the virus isolates of the avianAstrovirus according to the invention, or vice versa.

This demonstrated that the avian Astrovirus according to the inventionis a novel and unique serogroup of its own, which thus confirmed theunique molecular biological differences identified.

Plates with CEK cells were infected with isolate 19 Astrovirus, 3^(rd)passage, in serial 10 fold dilutions of the virus. After two days, theplates were fixed and stained with different antisera:

-   anti isolate 19 serum-pool from day 33 post inoculation, 1:20 in    PBS,-   anti ANV1 (strain SE-027/2), generated in chickens; 1:20 in PBS (the    ANV1 is a German ANV1 isolate, which upon DNA sequencing of specific    regions, appeared to be very closely related to the reference ANV 1    sequence from GenBank, that is used herein).-   anti CAstV2 (strain TS9L), generated in chickens, 1:500 in PBS (the    CAstV1 was derived from the sample that had been deposited at the    CNCM in Paris, France, under deposit number I-2932).-   negative control, only PBS.

After conjugation with goat anti-chicken IgG, the plates were read:

Virus Negative CEK Antiserum Isolate 19 ANV1 CAstV2 cells anti Isolate19 Positive Negative Negative Negative anti ANV1 Negative PositiveNegative Negative anti CAstV2 Negative Negative Positive Negativenegative Negative Negative Negative Negative

Example 12 Further Vaccination Trials Inactivated Vaccine:

Turkeys and chickens will be vaccinated with an adjuvated vaccine ofinactivated Astrovirus according to the invention, to optimise antigendose.

Astrovirus isolate 19 was cultured on CEL as descibed, and isinactivated using beta-propiolactone (BPL) under control of temperatureand pH, according to standard protocols. Inactivated Astrovirus ishomogenised into a standard w/o emulsion consisting of a light mineraloil and appropriate emulsifiers, using standard protocols.

Turkeys and chickens will be housed in isolated units: 120 two-week-oldconventional turkeys will be assigned to 6 separate groups (group 1-6)as they come to hand so that each group contains 20 animals. At threeweeks of age, the turkeys in group 1-4 will be vaccinated byintramuscular route with the w/o emulsion vaccine, containinginactivated Astrovirus isolate 19. The turkeys in group 5 will bevaccinated by subcutaneous route with the same W/O emulsion vaccine andthe turkeys in group 6 will not be vaccinated to serve as controls.

Similarly, 180 one-day-old SPF layer chickens will be assigned to 9separate groups (group 7-15) as they come to hand so that each groupcontains 20 chickens. At three weeks of age the chickens in group 7-10will be vaccinated by intramuscular route with a w/o emulsion vaccine,containing inactivated Astro type 3 virus. The chickens in group 11-14will be vaccinated with the same vaccine by subcutaneous route and thechickens in group 15 will not be vaccinated to serve as controls.

Before the start of the experiment, and at 4, 8, and 12 weekspost-vaccination blood samples will be taken from all turkeys andchickens. The sera will be examined for the presence of antibodiesspecific for the Astrovirus according to the invention and their titers,by IFT on CEL cells, or by VN assay.

This protocol can easily be modified to include a duration of immunitystudy if relevant; e.g. by keeping the vaccinated birds in isolators fora further 6-12 months and then apply a challenge infection with anAstrovirus according to the infection.

Live Vaccine:

Chickens will be vaccinated with a dose of live Astrovirus according tothe invention, and will subsequently be challenged with Astrovirus, tooptimise the route of application for a live Astrovirus vaccine.

100 one-day-old commercial MDA+ (maternally derived antibody positive)breeder chickens will be assigned to 5 separate groups so that eachgroup contains 20 chickens. At one day of age the chickens in group 1-4will be vaccinated with live Astrovirus isolate 19 from allantoic fluid,via eye-drop, coarse spray, aerosol spray, or drinking water. Thechickens in group 5 will not be inoculated to serve as unvaccinatedcontrols. At 4 weeks post-vaccination all chickens will be challengedwith an Astrovirus according to the invention. During three weeks afterchallenge all chickens will be observed daily for the occurrence ofclinical signs characteristic for Astrovirus infection and/or mortality.Twenty-one days post-challenge all remaining chickens will besacrificed, and submitted to histo-pathological examination.

1. An Avian Astrovirus having an open reading frame (ORF) 1a genomicregion, characterised in that the ORF1a of said avian Astrovirus, whencompared to the ORF 1a of avian nephritis virus 1, contains an insert of12 nucleotides, said insert being located in between nucleotidescorresponding to the nucleotides numbered 2485 and 2486 of SEQ ID NO: 1.2. The Avian Astrovirus according to claim 1, characterised in that theinsert of 12 nucleotides has a nucleic acid sequence as presented in SEQID NO:
 2. 3. The Avian Astrovirus according to claim 1, characterised inthat the ORF 1a of said avian Astrovirus comprises a region having anucleotide sequence identity of at least 88% with SEQ ID NO:
 4. 4. TheAvian Astrovirus according to claim 1, characterised in that from theORF 1a of said avian Astrovirus a PCR product of about 260 nucleotidescan be produced in a PCR-assay using a set of the primers represented inSEQ ID NO's: 30 and
 31. 5. The Avian Astrovirus according to claim 1,which is a virus as deposited under number CNCM I-3895 at the CollectionNationale de Cultures de Micro-organismes (CNCM), of the InstitutPasteur in Paris, France.
 6. An antibody or fragment thereof that canneutralise the avian Astrovirus of claim 5, in a virus neutralisationassay.
 7. An antigenic preparation obtainable from the avian Astrovirusaccording to claim
 1. 8. A DNA molecule comprising a region having anucleotide sequence identity of at least 88% with SEQ ID NO:
 4. 9. Aprotein comprising a region having an amino acid sequence identity of atleast 93% with SEQ ID NO:
 5. 10. A vaccine comprising the avianAstrovirus according to claim 1 and a pharmaceutically acceptablecarrier.
 11. The vaccine according to claim 10, comprising at least oneadditional antigen obtainable from a micro-organism pathogenic topoultry.
 12. A compound or a composition for use in a vaccine forpoultry, wherein the compound or composition is the avian Astrovirusaccording to claim
 1. 13. (canceled)
 14. The method for the manufactureof a vaccine for poultry, wherein a compound or composition is admixedwith an appropriate pharmaceutical carrier, and wherein the compound orcomposition is the avian Astrovirus according to claim
 1. 15. Adiagnostic kit comprising the avian Astrovirus according to claim
 1. 16.A vaccine comprising the antibody or fragment thereof according to claim6 and a pharmaceutically acceptable carrier.
 17. A vaccine comprisingthe antigenic preparation according to claim 7 and a pharmaceuticallyacceptable carrier.
 18. A vaccine comprising the DNA molecule accordingto claim 8 and a pharmaceutically acceptable carrier.
 19. A vaccinecomprising the protein according to claim 9 and a pharmaceuticallyacceptable carrier.